1. Stroma-Free Hemoglobin
Intravenously injected (infused) crude hemolysates and extensive hemolytic processes produced in vivo by immunological reactions involving intravascular lysis of red blood cells, are known to produce a clinical syndrome characterized by disseminate intravascular coagulation. This syndrome is often fatal and is produced by the residual red blood cell walls (stroma) and their fragments, so infused into circulating blood. Stroma-free hemolysates do not show this toxicity (See Rabiner et al, J. Exp. Med., .126:1127 (1967). As a result, it has been desired to use stroma-free hemoglobin as an oxygen carrier in cell-free transfusional fluids.
However, the use of stroma-free hemoglobin has the following two disadvantages: In vivo, the retention time of the stroma-free human hemoglobin is very short, i.e., it has a half-life on the order of 1-4 hours (see Rabiner et al, supra; and De Venuto et al, Transfusion, 17:555 (1977)). "Half-life" is defined as the time necessary to eliminate 50% of the infused hemoglobin from circulating blood. Further, outside of the red blood cells, hemoglobin has a high affinity for oxygen which, in vivo, would prevent the release, i.e., the transport, of oxygen from hemoglobin to the tissues. These disadvantages are directly the result of the molecular structure of hemoglobin.
Hemoglobin is a tetrameric molecule having a molecular weight of 64,500 Daltons. The tetrameric molecule is formed of two pairs of alpha and beta subunits. The subunits are held together as a result of ionic and Van der Waals forces, and not as a result of covalent bonds. When hemoglobin is oxygenated, i.e., combined with oxygen, it readily forms alpha-beta dimers having a molecular weight of 32,250 Daltons. These dimers are not retained in vivo by the kidneys and are eliminated through the urine.
The tetrameric structure of hemoglobin also provides a binding site for 2,3-diphosphoglycerate. Inside red blood cells, 2,3-diphosphoglycerate combines with hemoglobin in order to decrease its oxygen affinity to a level compatible with oxygen transport. The binding of 2,3-diphosphoglycerate and hemoglobin is purely electrostatic and no stable covalent bonds are formed. Thus, when red blood cells are ruptured and 2,3-diphosphoglycerate is not retained inside the cells by the cell wall, it is released from hemoglobin. As a result, hemoglobin acquires a higher oxygen affinity. This prevents the transport of oxygen from hemoglobin to the tissues. The level of this higher affinity is sufficient such that the oxygen affinity can be considered "non-physiological".
Because of the many appealing qualities of hemoglobin, i.e., its ability to reversibly bind oxygen, the low viscosity of a hemoglobin solution and its easy preparation and storage for long periods of time, various attempts have been made in order to overcome the above described disadvantageous characteristics of stroma-free hemoglobin. These various attempts are discussed in more detail below.
2. Chemical Treatments for Preventing the Formation of Dimers
The formation of alpha-beta dimers, which are not retained in vivo, can be prevented by coupling the tetrameric molecules of hemoglobin with large molecular weight matrices, ranging from 20,000 to 275,000 Daltons. For example, matrices such as dextran (see Tam et al, Can. J. Biochem., 56:981 (1978); and Bonneaux et al, Experientia, 37:884 (1981)) and hydroxyethyl starch (see Baldwin et al, Tetrahedron, 37:1723 (1981); and U.S. Pat. Nos. 4,412,989, 4,900,816, 4,650,786 and 4,710,488) have been employed. This coupling prevents the elimination of hemoglobin in vivo from the kidneys by way of the urine. Other types of polymeric coupling employing collagen, collagen degradation products, and gelatin as a supporting matrix have also been employed (see U.S. Pat. No. 2,591,133; U.S. Pat. No. 3,057,782; and Bowes et al, Biochem. Biophys. Acta., 168:341 (1968)). However, the oxygen affinity of the resulting coupled hemoglobin is even higher than that of stroma-free hemoglobin, and thus hemoglobin coupled in this manner cannot be advantageously employed as an oxygen transport medium.
Other known treatments for preventing the formation of alpha-beta dimers are based on reactions which polymerize the tetrameric molecules of hemoglobin to form so-called "polyhemoglobins". Polyhemoglobins can be obtained using bifunctional reagents, such as glutaraldehyde (see Hopwood et al, Histochem. J., 2:137 (1970)) or diimidate esters (see Mock et al, Fed. Proc., 34:1458 (1975); and U.S. Pat. No. 3,925,344). These bifunctional reagents form covalent bonds between the amino groups present on the surface of different hemoglobin molecules producing intermolecular cross-links. There are 40 or more of such amino groups belonging to lysyl residues on the surface of mammalian hemoglobins (44 in human hemoglobin). Thus, a large number of possible combinations of hemoglobin molecules occur. As a result, the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights thereof range from 64,500 to 600,000 Daltons. The separation of individual molecular species from the heterogeneous mixture is virtually impossible. In addition, although longer retention times in vivo are obtained using polyhemoglobins, the oxygen affinity thereof is higher than that of stroma-free hemoglobin.
Besides the various treatments discussed above which result in formation of heterogeneous mixtures of polyhemoglobin, reagents have been developed which are capable of producing an internal cross-link of the hemoglobin subunits with little or no formation of polyhemoglobins. More specifically, the formation of cross-links between the beta subunits of hemoglobin using 2-N-2-formylpyridoxal-5'-phosphate and borohydride and bis-pyridoxal polyphosphates have been carried out (see Benesch et al, Biochem. Biophys. Res. Comm., 62:1123 (1975); and Benesch et al, Biochem. Biophys. Res. Comm., 156:9 (1988)). The oxygen affinity of the thus treated hemoglobin is decreased to levels similar to that of normal blood. However, the reagents employed therein are very difficult and costly to synthesize, and thus these methods are disadvantageous.
Other reagents have been employed in order to effect internal cross-linking of the hemoglobin subunits. These reagents are commonly known as "diaspirins". Diaspirins are diesters of bis-3,5-dibromosalicyl containing succinyl, fumaryl or other dicarboxylic acid residues. These reagents produce covalent cross-links between two beta or two alpha subunits of an individual hemoglobin molecule. While better results are obtained using liganded (oxy- or carboxy-) hemoglobin, such a treatment does not sufficiently affect the oxygen affinity characteristics of stroma-free hemoglobin, and thus can not be advantageously employed (see Walder et al, J. Mol. Biol., 141:195 (1980); and U.S. Pat. Nos. 4,061,736, 4,001,200;, 4,001,401, and 4,053,590).
It should be stressed that according to pertinent literature, i.e., Walder, Biochemistry, 18:4265-4270 (1779); Walder et al, J. Mol. Biol., 141:195 (1980); and Zaugg, J. Biol. Chem., 255:2816-2821 (1980), activated dicarboxylic acid of increasing length, above that of the 4-carbon chain of succininc and fumaric acid, show a progressively lower reactivity with both liganded and unliganded hemoglobin so that activated sebacic acid (10-carbons long) produces no reactions with human hemoglobin.
In U.S. Pat. Nos. 4,473,496 and 4,857,636, linear alpha-omega or heterocyclic polyaldehydes containing negatively charged groups are described as suitable for both decreasing the oxygen affinity of hemoglobin and for producing inter- and intramolecular cross-linking of hemoglobin. These reagents include carbohydrate-containing molecules, such as raffinose, and mono- and polyphosphorylated nucleotides partially oxidized with periodate, so as to obtain aldehydic groups. The coupling reaction is based on the formation of Shiff bases of the aldehydic groups with the amino groups of the hemoglobin molecule. The Shiff bases are then transformed into covalent bonds by reduction with sodium or potassium borohydride, or another strong reducing agent.
In U.S. Pat. No. 4,584,130, cross-linking of hemoglobin with bifunctional reagents is disclosed. The reagents disclosed therein are based on an electron withdrawing group which modulates the reactivity of two peripheral active groups. However, the electron withdrawing group remains within the cross-linking bridge after the reaction. In the reagent of the present application, when an electron withdrawing group is employed, it is present in the leaving group only (e.g., 3,5-dibromosalicyl), and therefore it does not remain in the cross-linking bridge after the chemical reaction of the activated carboxyls with the amino groups of the protein.
3. Chemical Treatments for Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
The most widely used chemical modification of stroma-free hemoglobin so as to decrease the oxygen affinity thereof employs the use of pyridoxal-5'-phosphate and sodium or potassium borohydride (see Bensch et al, Biochem., 11:3576 (1972)). The resulting product is commonly referred to as "PLP-hemoglobin" and has satisfactory oxygen affinity, i.e., oxygen affinity very near that of the red cells present in normal blood.
Other known chemical modifications of hemoglobin have been carried out using phosphoric acid derivatives of carbohydrates (e.g., glucose-6-phosphate) (see McDonald et al, J. Biol. Chem., 254:702 (1979)); carbamylation (see Manning, Meth. Enz., 76:159 (1981)) and carboxymethylation (see DiDonato et al, J. Biol. Chem., 258:11890 (1983)). In each of these treatments, the amino-terminal end of the beta subunit of hemoglobin is permanently substituted with the above described reagents.
In addition, none of these chemical treatments discussed in this section stabilize the tetrameric structure of hemoglobin so as to prevent the formation of alpha-beta dimers. Thus, the resulting hemoglobins do not have prolonged retention times in vivo.
4. Combined Chemical Treatments for Preventing the Formation of Alpha-Beta Dimers and Decreasing the Oxygen Affinity of Stroma-Free Hemoglobin
As discussed above, the production of physiologically competent stroma-free hemoglobin-based oxygen carriers necessitates two separate treatments. That is, one treatment is necessary for preventing the formation of alpha-beta dimers in vivo and a second treatment is required for decreasing its oxygen affinity. The most widely employed combination of treatments is that of reacting glutaraldehyde with PLP-hemoglobin to form pyridoxylated polyhemoglobins (see Seghal et al, J. Surg. Res., 30:14 (1981). Intramolecular cross-linking of PLP-hemoglobin has also been obtained using diaspirins (see Tye et al, Prog. Clin. Biol. Res., 22:41 (1983)).
It should be noted that only stroma-free hemolysates or washed red blood cells are utilized in the above-cited articles. That is, purification procedures for isolating the hemoglobin component of the stroma-free hemolysates are not described therein. Thus, what is defined as stroma-free hemoglobins therein is in actuality stroma-free hemolysates.
More specifically, about 95% of the hemolysate components is hemoglobin. The remainder consists of proteins and polypeptides whose pharmacological and immunological toxicity is not known. When used for infusion in animals, several grams of hemolysate-containing hemoglobin are injected. Thus, undesirably, hundreds of milligrams of substances of unknown biological activity are also infused into animals when employing a hemolysate.
It should also be noted that in the above-cited references, purification procedures for isolating the desired hemoglobin products from the reaction mixture are not described therein. It is impossible to avoid the presence of overreacted and underreacted hemoglobins in the reaction mixtures. These products do not have the desired functional and molecular characteristics.
For the above reasons, it is advantageous to perform chemical treatments on purified hemoglobins, and then to purify the product of the reaction.
The reagents of the present invention present clear advantage over previously employed divalent reagents for producing intramolecular cross-linked hemoglobin. Specifically, using the present cross-linking reagents, the resulting cross-linked hemoglobin of the present invention has a lower oxygen affinity, and can be obtained in a much higher yield. While the cross-link is still intramolecular, the hemoglobin of the present invention has been found to be stable, not only against dissociation, but also against physical agents like heat, pH and aging. Thus, the formation of ferric hemoglobin is greatly retarded with the cross-linked hemoglobin of the present invention. This allows the use of high temperature heat treatments for eliminating pathogens, the use of lyophilization procedures and to effect storage in liquid form in the cold and at room temperature.